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1521-0103/360/2/346–355$25.00 http://dx.doi.org/10.1124/jpet.116.237313THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 360:346–355, February 2017Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Pharmacological Characterization of a Novel Beta 3 AdrenergicAgonist, Vibegron: Evaluation of Antimuscarinic ReceptorSelectivity for Combination Therapy for Overactive Bladder s

J. Di Salvo, H. Nagabukuro, L. A. Wickham, C. Abbadie, J. A. DeMartino, A. Fitzmaurice,L. Gichuru, A. Kulick, M. J. Donnelly, N. Jochnowitz, A. L. Hurley, A. Pereira, A. Sanfiz,G. Veronin, K. Villa, J. Woods, B. Zamlynny, E. Zycband, G.M. Salituro, T. Frenkl,A. E. Weber, S. D. Edmondson, and M. StruthersDiscovery and Preclinical Sciences, Merck Research Laboratories, Kenilworth, New Jersey

Received August 17, 2016; accepted December 5, 2016

ABSTRACTAlthough the physiologic role of muscarinic receptors in bladderfunction and the therapeutic efficacy of muscarinic antagonistsfor the treatment of overactive bladder are well established, therole of b3-adrenergic receptors (b3ARs) and their potential astherapeutics is just emerging. In this manuscript, we character-ized the pharmacology of a novel b3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of b3ARagonism and muscarinic antagonism in urinary bladder function.Vibegron is a potent, selective full b3AR agonist across species,and it dose dependently increased bladder capacity, decreasedmicturition pressure, and increased bladder compliance inrhesusmonkeys. The relaxation effect of vibegronwas enhancedwhen combined with muscarinic antagonists, but differentiallyinfluenced by muscarinic receptor subtype selectivity. The effectwas greater when vibegron was co-administered with tolterodine,

a nonselective antagonist, compared with coadministration withdarifenacin, a selective M3 antagonist. Furthermore, a synergisticeffect for bladder strip relaxation was observed with the combi-nation of a b3AR agonist and tolterodine in contrast to simpleadditivity with darifenacin. To determine expression in rhesusbladder, we employed a novel b3AR agonist probe, [

3H]MRL-037, that selectively labels b3 receptors in both urothelium anddetrusor smooth muscle. Vibegron administration caused adose-dependent increase in circulating glycerol and fatty acidlevels in rhesus and rat in vivo, suggesting these circulatinglipids can be surrogate biomarkers. The translation of our obser-vation to the clinic has yet to be determined, but the combination ofb3AR agonists with M2/M3 antimuscarinics has the potential toredefine the standard of care for the pharmacological treatment ofoveractive bladder.

IntroductionThe beta-adrenergic receptor (bAR) family was first de-

scribed more than 60 years ago (Ahlquist, 1948) and has beensince divided into three subtypes: b1, b2, and b3 (Lands et al.,1967; Emorine et al., 1989). For the b3AR subtype, tissueexpression is more restricted compared with b1 and b2, withadipose, heart/vasculature, urinary bladder (mRNA) andovary (protein) believed to have the highest expression(Thomas and Liggett, 1993; Berkowitz et al., 1995; Uhlénet al., 2015). In the urinary bladder detrusor muscle ofmammals, all three bAR subtypes mRNA are expressed(Nomiya and Yamaguchi, 2003). Additionally, significantexpression of b3AR protein is observed in bladder urothelium(Limberg et al., 2010), the luminal epithelial lining of theurinary bladder (Birder and de Groat, 2007).

The urinary bladder functions to collect and store urineexcreted by the kidneys until voided. Alternating phases ofcontinence and micturition are controlled by the interplay ofthe central and peripheral nervous system with the localrelease of regulatory agents (Andersson and Wein, 2004;Beckel and Holstege, 2011). Bladder filling occurs by relaxa-tion of the detrusor muscle (via parasympathetic inhibition)with simultaneous contraction of the urethral sphincters toprevent involuntary emptying. It is also thought that releaseof norepinephrine from the sympathetic hypogastric nerveresulting in activation of primarily bAR receptors enhancesbladder compliance via detrusor relaxation, although onlysparse sympathetic innervation is observed in the bladderdome (Fowler et al., 2008). Bladder emptying (so-calledmicturition) on the other hand occurs via a switch in efferentsignaling from sympathetic to parasympathetic, resulting in arelease of acetylcholine and to a lesser extent ATP from thepelvic nerve causing detrusor contraction and interruption ofrelease of norepinephrine from the hypogastric nerve, causingurethral sphincter relaxation. Acetylcholine primarily acts onthe detrusor via muscarinic M2 and M3 subtypes, whereas

J.D.S and H.N. contributed equally to this work.dx.doi.org/10.1124/jpet.116.237313.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: AE, adverse effects; bAR, beta-adrenergic receptor; b3AR, b3-adrenergic receptor; CIx, combination index; CI, confidenceinterval; EFS, electrical field stimulation; FFA, free fatty acid; OAB, overactive bladder.

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ATP acts on P2X-purine receptors, particularly under patho-logic conditions (Andersson, 2015), in the detrusor to initiatebladder contraction.Disruption of this coordinated communication can result in

a symptom complex characterized by urinary urgency, with orwithout urgency-associated urinary incontinence, referred toas overactive bladder (OAB) (Abrams et al., 2002). It isestimated that 12–23% of the general population exhibitssymptoms of OAB, with a significant degradation in quality oflife (Irwin et al., 2006; Coyne et al., 2011). Management ofOAB is typically a multimodal approach, employing bothnonpharmacological and pharmacological treatment para-digms. When nonpharmacological approaches (i.e., lifestyleand dietary modification) are ineffective, antimuscarinics arethe first-line pharmacological treatment, representing themost commonly prescribed drug therapy (Abrams andAndersson, 2007). Although antimuscarinics have beendemonstrated to improve urgency and decrease frequency ofmicturition and urge incontinence, blockade of the M3muscarinic receptor can lead to adverse effects (AEs) suchas dry mouth and constipation (Abrams and Andersson,2007). These AEs contribute to a high discontinuation rate,resulting in significant unmet medical need for a pharmaco-logical treatment of OAB with an improved AE profile whilemaintaining or exceeding the efficacy of antimuscarinics(Chapple et al., 2008).b3AR activation in the bladder represents themost relevant

mechanism to increase bladder capacity without affectingbladder contraction. Initial studies in isolated human bladderusing nonselective bAR agonists such as isoproterenol dem-onstrated a pronounced bladder relaxation (Anderson, 1993).Subsequent studies using selective b3AR agonists in isolatedhuman detrusor muscle strips determined that the observedrelaxation was due to activation of b3AR (Rouget et al., 2014;Gillespie et al., 2015; Michel and Korstanje, 2016). Givenincreasing evidence for b3AR activation as a treatment ofOAB, efforts to discover or repurpose potent and selectiveb3AR agonists were initiated in recent years (Furuta et al.,2006; Drake, 2008). Subsequently, four selective b3AR ago-nists, mirabegron (Tyagi and Tyagi, 2010), ritobegron(Maruyama et al., 2012), solabegron (Ohlstein et al., 2012),and vibegron (Edmondson et al., 2016) entered clinical trialsfor treatment of OAB, with published clinical data availablefor both mirabegron and solabegron. In 2012, mirabegrondemonstrated significant efficacy in reducing micturitionfrequency, urgency incontinence, and increasingmean volumeper micturition in patients with OAB (Chapple et al., 2014)and subsequently received regulatory approval.Herein, we describe the pharmacological characterization of

the novel b3AR agonist vibegron (MK-4618, KRP-114V)(Edmondson et al., 2016) currently under clinical developmentfor OAB. In addition, we describe the mechanistic interactionof b3AR agonists and antimuscarinics in bladder function andwe propose an optimal combination of these mechanisms inthe treatment of OAB.

Materials and MethodsSubjects. All procedures related to the use of animals were

approved by the Institutional Animal Care and Use Committee atMerck Research Laboratories (Rahway, NJ) and conform to the NIHGuide for the Care and Use of Laboratory Animals (8th edition, 2011).

A total of 16 adult female rhesus monkeys (Macaca mulatta) weighing5 to 7 kg and 4–7 years of agewere used. Animals were either paired orindividually housed in temperature/humidity controlled rooms on a12-hour light/12-hour dark cycle and fed standard laboratory chow(Tekland, Harlan Laboratories, Indianapolis, IN) supplemented withfresh fruit and vegetables with ad libitum water. A total of 81 maleadult Sprague-Dawley rats weighing 250 to 350 g (Charles River,Wilmington, MA) were housed in temperature and light-controlled(12-hour light/12-hour dark cycle) cages, fed standard laboratory chow(Diet 7012, Harlan) and water ad libitum.

Reagents. Vibegron ((6S)-N-[4-({(2S,5R)-5-[(R)-hydroxy(phenyl)methyl]pyrrolidin-2-yl}-methyl)phenyl]-4-oxo-4,6,7,8-tetrahydropyrrolo[1,2-a]pyrimidine-6-carboxamide), MRL-037 ((R)-2-amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro-4H-cyclopenta[d]thiazole-4-carboxamide), and [3H]MRL-037 ((R)-2-amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl-3,5-t2)methyl)pyrrolidin-2-yl)methyl)phenyl-2,6-t2)-5,6-dihydro-4H-cyclopenta[d]thiazole-4-carboxamide) were synthesized by theDiscovery ChemistryDepartment(Merck Research Laboratories) as previously described (Moyes et al.,2014; Edmondson et al., 2016; Supplemental Fig. S1). CL316,243(disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-propyl]-1,3-benzodioxole-2,2-dicarboxylate hydrate), oxybutynin chlo-ride, andmethoctramine hydrate were purchased from Sigma-Aldrich(St. Louis, MO). Tolterodine L-tartrate and darifenacin hydrobromidewere purchased from Toronto Research Chemicals (North York,Canada) and AK Scientific (Union City, CA), respectively.

Autoradiography of Bladder Sections with [3H]MRL-037—ExVivo Receptor Occupancy Assay. Rhesus monkeys wereanesthetized with an intramuscular injection of 5 mg/kg Telazol(Zoetis, Parsippany, NJ) or ketamine HCl (20 mg/kg) followed by alethal dose of a euthanasia drug (i.e., Euthasol; Virbac AH Inc., FortWorth, TX). The bladder was then surgically removed and cut into20-mm-thick sections, collected on superfrost/plus slides using arapid sectioning cryostat (Leica CM1900), and then air dried for30–60 minutes at room temperature. After marking boundaries aroundthe section with pan-pep, the tissue was covered with 600–1000 ml ofbinding buffer (50 mMTris-HCl, 2 mMMg2Cl, 1 mMCaCl2, 5 mMKCl,0.1% bovine serum albumin) containing 50 nMof [3H]MRL-037 (specificactivity of 30-50 Ci/mmol; Merck Research Laboratories) without orwith 10 mM of cold MRL-037 for nonspecific binding for 25 minutes.After incubation, the slides were washed for 4 minutes with ice-coldbinding buffer containing 0.05% TritonX-100 for total of six times andthen soaked twice into in 30ml of ice-cold bufferwith 0.05%TritonX-100in a staining jar for 4 minutes each wash, followed by dipping the slidesthree times in ice-cold water. Slide were dried and exposed to KodakMR-2 8952855SigmaKodakBioMaxMR film (Cat #Z350400-50EA) for5 days. Adjacent bladder sections were stained with hematoxylin andeosin for histology following standard protocol (Longnecker, 1966).

In Vitro Potency and Selectivity of Vibegron on b-AdrenergicReceptors. The ability of vibegron to activate the human, rhesus,rat, and dog b1, b2, and b3AR was measured using CHO cell linesstably expressing the appropriate adrenergic receptor (Candeloreet al., 1999). For b3AR, the human cell line used expressed b3 atlevels similar to those observed in human detrusor muscle (Supple-mental Fig. S2). To quantify the amount of released cAMP after b-ARactivation, the LANCE cAMP kit (Perkin Elmer, Shelton, CT), a time-resolved fluorescence resonance energy transfer immunoassay, wasused. Compounds were serially diluted in DMSOand an aliquot addedto either 384-well or 96-well micro titer plates in assay buffer (5 mMHEPES, 0.1%bovine serumalbumin inHank’s balanced salt solution).The reaction was initiated by the addition of 6000 cells per well inassay buffer that also contained a cAMP-specific antibody labeledwithAlexa Fluor 647 and a phosphodiesterase inhibitor (IBMX, Sigma). Toexamine serum-shifted potency, efficacy was evaluated using an assaybuffer containing 40% pooled human, rhesus, or rat serum. Because ofa smaller assay window with serum compared with the buffer assay,the number of cells was increased to 10,000 per well. After 30-minuteincubation at room temperature, the cells were lysed by the addition of

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LANCE detection buffer containing a europium-labeled cAMP tracer.Fluorescence was measured after 1-hour incubation at room temper-ature using a PE Envision reader, exciting at 340 nm and measuringemission at 615 and 665 nm. For each assay, a cAMP standard curvewas included and used to convert fluorescence readings directly tocAMP amounts. Values were normalized to a known full agonist(isoproterenol), and the EC50 and percent maximum activation wasthen determined.

Isometric Detrusor Muscle Tension Recordings. Rats wereeuthanizedwithCO2 and the entire bladderwas surgically removedandthe mucosa was left intact. The bladder was then placed in a bath ofKrebs solution (113 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mMMgSO4, 25 mM NaHCO3, 1.2 mM KH2PO4, and 1.5 mM glucose). Thebladder domewas dissected in four approximately equal pieces of about6 mm � 3 mm. Each strip was placed in a warmed (37°C) organ bath(25 ml) containing oxygenated (95% O2 1 5% CO2) Krebs solution. Thestrips were tied at one end to the organ bath and connected at the otherend to an isometric force transducer (AD Instruments, ColoradoSprings, CO) under a resting tension of 10 mN. The responses of thepreparations were recorded by a multiple channel data acquisitionsystem (PowerLab, AD Instruments), and measured with an analysissoftware (Chart 5, AD Instruments). After the equilibration period for atleast 60 minutes, each tissue strip was challenged to electrical fieldstimulation (EFS) at 60 Hz; duration, 0.3 ms; 3 seconds; 90 V to inducecontractions. Upon obtaining stable contractions with EFS, i.e., base-line, compound solution (25 ml) was applied into the organ bath in acumulative manner, followed by another EFS (consecutive 3 stimula-tions with 2-minute intervals) 15 minutes after compound treatment.Average amplitude of three contractions at baseline and each concen-tration, changes from baseline, and then ratio to corresponding vehiclegroup (% of control) were calculated.

Isobologram Analysis for Assessment of Combined DrugEffects. Isobologram analysis (Tallarida, 2001) was used to deter-mine whether the interactions between a selective b3AR agonistCL316,243 and antimuscarinic drugs were additive, synergistic, orantagonistic. CL316,243 was used in this study, because this com-pound was known to be potent at rat b3AR. Dose-dependent effectswere determined for each compound and for combinations at fixedconstant ratios. Data points on the isobologram were evaluatedaccording to their positions relative to the diagonal. Data points inthe lower left region indicated synergism, falling on the diagonal lineindicated additive effects, and the upper right region indicatedantagonism. The combination index (CI) provided a means to analyzethe combined effects with a median-effect plot analysis. The CI wascalculated according to the following formula: CI 5 (dCL/IC25CL) 1(dA/IC25A), where IC25CL is the concentration of CL316,243 requiredto produce 25% inhibition versus control, and dCL is the concentrationof CL316,243 required to produce 25% inhibition when combined withan antimuscarinic drug. Similarly, IC25A is the concentration of anantimuscarinic required to produce 25% inhibition, and dA is theconcentration of an antimuscarinic required to produce 25% of theeffect when combined with CL316,243. The CI values were defined asfollows:,0.85 synergism, from 0.8 to 1.2 5 additive effect, and.1.25 antagonism. The IC25 values and their 95% confidence intervalswasinterpolated from the nonlinear fit curve (Prism 5, GraphPad, LaJolla, CA).

Cystometry in Rhesus Monkeys. Cystometry was performed asdescribed previously (Nagabukuro et al., 2011). In brief, rhesusmonkeys were anesthetizedwith an intramuscular injection of Telazol(5 mg/kg) or ketamine HCl (20 mg/kg) followed by intravenousconstant rate infusion with ketamine HCl (0.2–0.3 mg/kg/min).Animals were then placed in a supine position, and two catheters(20 gauge) were inserted into a saphenous and/or a brachial cephalicvein for compound administration and ketamine infusion/bloodsampling, respectively. A triple lumen balloon transurethral catheter(7.4 Fr, CookMedical, Bloomington, IN) was inserted into the bladder,and the balloon was inflated with 1 ml of saline to secure the catheterat the bladder base. Another two lumens were connected to a pressure

transducer and an infusion pump for intravesical pressure measure-ment and intravesical saline infusion, respectively. The intravesicalpressure was recorded using a multiple channel data acquisitionsystem (Power Laboratory 4/30, AD Instruments) at a sampling rate of20 Hz. After confirming bladder emptiness by ultrasonography, salinewas intravesically infused at 15 ml/min. Saline infusion was discon-tinued when a rapid increase in the intravesical pressure due to themicturition reflex was observed. After two baseline cystometryreadings, a compound was intravenously dosed using a rising doseparadigm, with a cystometry measurement performed 10 minutesafter each dose. Blood samples were taken for measurements ofplasma compound levels and serum glycerol/free fatty acid (FFA)levels right after each cystometry. The following cystometric param-eters were obtained from each cystometry: bladder capacity (durationof bladder filling multiplied by intravesical infusion rate), maximummicturition pressure (the pressure reading of the first peak in intra-vesical pressure driven by the micturition reflex), and bladdercompliance (inverse of average slope of intravesical pressure duringfilling phase). The changes from baseline were calculated andcompared with the values in the vehicle-treated group or baselinevalues. Compounds were dissolved in sterile 60% PEG400-20%ethanol-20% saline in a volume of 0.2 ml/kg. Serum glycerol levelswere determined using commercially available kits (Sigma). Plasmaconcentrations of compoundswere determinedby liquid chromatography-tandem mass spectrometry on an Applied Biosystems API 4000 massspectrometer. Plasma glycerol and FFA levels were determined usingcommercially available kits (FG0100, Sigma and NEFA-HR2, Wako,Richmond, VA; respectively) which rely on a series of enzyme coupledreactions.

Statistical Analysis. Data are presented as arithmetic mean 6S.D., S.E.M., or geometric mean with confidence intervals (CI) exceptwhere indicated. The mean values were compared with two-wayanalysis of variance with Bonferroni’s post hoc test or one-way analysisof variancewithDunnett’smultiple comparison test. A probability valueless than or equal to 0.05 was considered significant.

ResultsPotency of Vibegron at b-Adrenergic Receptors

across Species. Vibegron (Fig. 1A) potently activates humanb3AR and increases cAMP levels, with an EC50 of 1.1 nM and87% activation relative to isoproterenol (Edmondson et al.,2016). Vibegron is also highly selective over b1AR and b2ARversusb3ARacrossmultiple species, demonstrating.9000-foldselectivity for activation of b3AR over b1AR or b2AR in cellbased in vitro functional assays (Table 1). A small serum shiftwas observed in the presence of 40% human serum (EC501.7 nM; Fig. 1B). The small effect of serumwas also observed forrat and rhesus b3AR, consistent with the low noncovalentplasma protein binding (Edmondson et al., 2016).Localization of b3AR in Rhesus Bladder with a

Labeled b3AR Agonist. To determine expression of b3ARin rhesus tissue, we used a radiolabeled agonist of b3AR,[3H]MRL-037 (Fig. 2A; Supplemental Fig. S1), a pan-speciespotent and selective b3AR agonist (Moyes et al., 2014), intissue autoradiography experiments. This compound is apotent b3AR agonist across multiple species with no activityat b1 or b2 and with physiochemical properties that make it asuitable tracer for localizing b3AR expression in tissue.Similar to the staining pattern in human tissue observedwith immunohistochemistry (Limberg et al., 2010), clearstaining of the urothelium was observed in rhesus bladderusing [3H]MRL-037 (Fig. 2, B and C). Staining of humanbladder tissue with 3H-MRL-037 produced a similar pattern,

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albeit with weaker signal, than was observed in rhesus(Supplemental Fig. S3).Dose-dependent Effects of Vibegron in Urodynamic

Parameters in Rhesus Monkeys. We previously reportedthe activity of vibegron in a rat bladder hyperactivity model(Edmondson et al., 2016), measured by cystometry, and thecomparison with activity in adipose tissue as measured bylipolysis readouts. We sought to extend the evaluation ofvibegron to nonhuman primates. A nonhuman primate phar-macodynamic model offers advantages for pharmacologicalevaluation of vibegron because of physiologic and anatomicsimilarities of the lower urinary tract to humans. Additionally,vibegron activates both human and rhesus monkey b3AR at asimilar potency (Table 1). Accordingly, vibegron was evaluatedacross concentrations up to 10 mg/kg i.v. in the rhesuscystometry model. As investigated in the rat bladder

hyperactivity model, a relationship between indices of bladderfunction and target engagement markers of acute b3ARstimulation in adipose tissue, i.e., circulating glycerol increasein rhesus monkeys, was also examined. Baseline values ofurodynamic parameters in vehicle- and vibegron-treated ani-mals are as follows: bladder capacity, 156.86 21.6 ml (vehicle),123.76 10.7 ml (vibegron); micturition pressure, 36.76 1.6 cmH2O (vehicle), 35.5 6 2.8 cm H2O (vibegron); bladder compli-ance, 15.0 6 2.2 ml/cm H2O (vehicle), 16.6 6 2.8 ml/cm H2O(vibegron). As in the previous study (Nagabukuro et al., 2011),vehicle had no statistically significant effect on any of param-eters. Vibegron increased bladder capacity in a dose-dependentmanner (Fig. 3A). The maximum bladder capacity increaseinduced by vibegron was 156% of baseline value, whichis comparable to maximum effect with antimuscarinics(Nagabukuro et al., 2011). Micturition pressure was signifi-cantly decreased at 0.3 and 3 mg/kg (Fig. 3B). Bladdercompliance was increased at doses greater than 0.1 mg/kg(Fig. 3C). Vibegron also increased serum glycerol and FFAlevels in a dose-dependent manner (Fig. 3, D and E). Concen-tration response curves for bladder capacity and serum glycerolclosely overlapped (see Table 2 for vibegron plasma levels),where theEC50 value in increasing bladder capacitywas 2.9 nMin total plasma and 1.5 nM at unbound levels; for increasingserum glycerol, the EC50 value was 9.9 nM in total plasma and5.0 nM at unbound levels (Edmondson et al., 2016).Effect of Combined Treatments of a b3AR Agonist

with Antimuscarinics in Isolated Detrusor Muscle.CL316,243 and all antimuscarinics oxybutynin, tolterodineand darifenacin, inhibited the EFS-induced isolated detrusormuscle contractions in a concentration-dependent manner(Fig. 4, A and B). Unlike oxybutynin, vibegron also depressedthe spontaneous contractile activity. Concentrations thatinduced 25% inhibition (IC25) were used to determinecombination ratios and for isobologram analyses (Table 3).CL316,243 was cotreated with tolterodine, oxybutynin, ordarifenacin at a fixed combination ratio. Isobologram anal-yses are shown in Fig. 5. Based on the CI criteria (seeMaterials and Methods), all combinations were synergistic,but with different degrees of synergism: tolterodine .

TABLE 1Potency and selectivity of vibegron at human, rhesus, rat, and dog b1, b2, and b3ARPotency was measured using CHO cell lines stably expressing the appropriate receptor (Bmax 50–100 fmol/mg for b3ARcell lines, 300–500 fmol/mg for b1 and b2). To determine potency in the presence of serum, compounds were assayed in thepresence of 40% autologous serum (pooled donors). EC50 for vibegron in the presence of 40% dog serum was notdetermined. Activation was determined relative to the known full agonist isoproterenol (1 mM). EC50 of isoproterenol atthe human, rhesus monkey, rat, and dog b3AR receptors was 28, 12, 124, and 264 nM, respectively. Values representgeometric mean with confidence intervals (CI); N $ 6 for all values. b3AR potency has been previously described(Edmondson et al., 2016).

bAR Subtype Species EC50 (CI) EC50, nM w/ 40% Serum (CI)

nM % activation

b3 Human 1.0 (1.5,0.71); 84% 1.5 (2.3, 1.0); 102%b1 Human .10,000; 5%b2 Human .10,000; 7%b3 Rhesus Monkey 0.52 (0.81, 0.30); 108% 5.5 (12, 2.6); 98%b1 Rhesus Monkey .10,000; 4%b2 Rhesus Monkey .10,000; 0%b3 Rat 81 (119,55); 83% 118 (161,85); 89%b1 Rat .10,000; 0%b2 Rat .10,000; 1%b3 Dog 10 (15, 7.1); 82% N.D.b1 Dog .10000; 2%b2 Dog .10,000; 1%

N.D., not determined.

Fig. 1. (A) Structure of vibegron; (B) activation of recombinant humanb3AR by vibegron in the absence (s) or the presence (d) of 40% humanserum assayed in a recombinant CHO cell line that stably expresseshuman b3AR. Percent activity is an average ofN = 3 determinations; errorbars represent standard deviation. Potency of vibegron (1.3 nM) is onlyslightly less potent (1.7 nM) when assayed in the presence of 40% humanserum.


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oxybutynin. darifenacin (Fig. 5). TheM2 selective antagonistmethoctramine caused only marginal inhibition of the EFS-induced detrusor contraction (0.003–10 mM; SupplementalFig. S4). Given the potency of methoctramine at muscarinicreceptor subtypes other than M2 (Ki, ∼1 mM), significantinhibition at the higher concentrations was expected. But atleast in the current experimental setting, there was less than25% inhibition of EFS-induced isolated detrusor contraction.In addition, pretreatment of methoctramine (1 mM) did notaffect CL316,243-induced inhibition of detrusor contraction(data not shown). However, methoctramine caused a signifi-cant change in isobologram for the darifenacin and CL316,243combination. As shown in Fig. 5D, combination withCL316,243 and darifenacin exhibited much more robustsynergism with a pretreatment of methoctramine (CI, 0.16)compared with the same 1 mM).Effect of Combined Treatments of Vibegron and

Antimuscarinics in Rhesus Monkeys. To extend ourfindings of combination therapy with vibegron, cotreatmentsof vibegron and two antimuscarinic agents tolterodine anddarifenacin were evaluated in the cystometry model in rhesusmonkeys. We used monotherapy data for tolterodine anddarifenacin from our previous publication (Nagabukuroet al., 2011) as we carried out the study with exactly the samemethods and within similar timeframe. All dose combinationsof vibegron and tolterodine showed a greater bladder capacityincrease compared with each compound alone, with effectsthat are greater than additive at low doses (Table 4). Incontrast, addition of darifenacin to vibegron induced greaterbladder relaxation in rhesus only when used at high doses.

DiscussionGiven the questionable selectivity of antibodies for staining

of b3AR in rhesus monkeys, we turned to a more novelapproach of identifying b3AR expression in the urinarybladder of this species—use of a selective b3 agonist

radioligand, MRL-037—to label b3AR expressing cells in theurinary bladder. From our in vitro characterization of MRL-037, this compound is extremely selective for b3 across severalspecies, including rhesus monkeys. Using autoradiography ofbladder sections incubated with [3H]MRL-037, b3AR expres-sion was detected in urothelium. Although less intense, b3ARexpression was detected in detrusor as well, similar to whatwas previously observed in human bladder, where b3ARexpression appears to be more intense in the urotheliumcompared with the detrusor (Limberg et al., 2010). Thepotency and selectivity of MRL-037 across several speciesprovides for a unique means to directly compare and quantifyb3AR expression across multiple species using a single tool. Inaddition, MRL-037 could be employed to determine ex vivoreceptor occupancy, providing a means to directly correlateb3AR target engagement in the bladder to efficacy.We observed staining for b3AR in the urothelium of human

and rhesus monkey. Given our data, along with previouslypublished observations (Limberg et al., 2010; Kullmann et al.,2011; Otsuka et al., 2013), b3AR is expressed in both thedetrusor muscle and bladder urothelium across multiplespecies. The exact role of b3AR in the urothelium and itscontribution to the observed efficacy of b3AR agonists intreating OAB is unknown, but there is mounting evidencethat b3ARs in the urothelium likely contribute either directlyor indirectly to the observed clinical effects of b3AR agonists.Our demonstration of agonist binding to urothelium and thepreviously reported b3AR agonist-induced functionality in animmortalized human urothelium cell line (Harmon et al.,2005) infer that b3AR agonists can have a direct effect onactivating cells within the urothelium. However, the func-tional role of b3ARs in the urothelium has not been welldocumented. Masunaga et al. (2010) and Kullmann et al.(2011) showed that in porcine and rat bladder strips re-laxation of the detrusor muscle by nonselective bAR andselective b3AR stimulation occurs to the same extent with orwithout urothelium. Alternatively, others reported that the

Fig. 2. MRL b3AR Agonist [3H]MRL-037

(A) is a potent full agonist at the human(EC50 = 0.12 nM), rhesus (EC50 = 0.12nM), rat (EC50 = 1.0 nM), and dog (EC50 =0.19 nM) b3ARs with no affinity for the b1and b2 subtypes (EC50 and IC50 . 10 mMall species). Comparison of binding of[3H]MRL-037 to histologic staining inrhesus bladder tissue: (B) autoradiogra-phy after incubation of bladder with 50 nM[3H]MRL-037; (C) hematoxylin and eosinstaining; (D) to determine nonspecificbinding, incubation of bladder with50 nM [3H]MRL-037 and an excess(10 mM) of unlabeled MRL-037 wasperformed followed by autoradiogra-phy. Arrows indicate regions of intacturothelium. Adjacent sections of blad-der are shown. Data are representativeof three studies.

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presence of urothelium diminishes nonselective bAR-inducedrelaxation of isolated human detrusor muscle (Otsuka et al.,2008; Propping et al., 2013). By using subtype selective bARantagonists, b2AR was suggested to be involved in this

urothelial effect, whereas b3AR directly mediates the relaxa-tion of human detrusor, and its involvement did not differ withor without urothelium (Propping et al., 2013).Medicinal chemistry efforts at optimizing the potency,

selectivity, and pharmacokinetic properties of a series ofpyrrolidine derived amides led to the discovery of vibegron,which is currently in late stage clinical trials for OAB(Edmondson et al., 2016). In two different preclinical species,vibegron causes dose-dependent relaxation of urinary bladder,resulting in an increase in bladder capacity and a decrease inmicturition pressure. These pharmacodynamic effects in thelower urinary tract accompany an increase in circulatingglycerol and FFA, indicating the potential use of b3ARactivation in adipose tissue as a surrogate pharmacodynamic/target engagement readout for urinary bladder. Interpretationof the relative pharmacodynamics effects of b3AR activation inadipose tissue to bladder must be done with care when trans-lating across organs and species. In rhesus cystometry studies,

Fig. 3. Effect of vibegron on the bladder capacity (A), maximum intravesical pressure (B), bladder compliance (C), serum glycerol (D), and nonesterifiedfree fatty acids (NEFFA) (E) in anesthetized rhesus monkeys. Values represent mean 6 S.D. of value or of percentage of baseline values as indicated. N = 6.*P , 0.05, **P , 0.01 versus baseline values, repeated-measures analysis of variance; #P , 0.05, ##P , 0.01, versus vehicle-treated group, Dunnett’smultiple comparison test. a, Not applicable for statistical analyses because of lack of data from one animal (N = 5).

TABLE 2Plasma levels of vibegron in anesthetized rhesus monkeysValues represent mean 6 S.D. (N = 4–6).

Plasma Vibegron Level

nM

0.003 mg/kg 0.5 6 0.40.01 mg/kg 1.2 6 0.10.03 mg/kg 3.0 6 0.90.1 mg/kg 13.1 6 3.60.3 mg/kg 43.4 6 15.41 mg/kg 128.5 6 27.93 mg/kg 553.6 6 144.0


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elevated levels of serum glycerol correlated with b3AR-mediated increases in bladder capacity, with the two readoutsof b3AR activation closely overlapping each other. In rats,although decreases inmicturition pressure are accompanied byan increase in circulating glycerol levels, we observed elevatedglycerol levels in the absence of any effect on bladder compli-ance, suggesting that rat adipose is the more sensitive tissue tob3AR activation relative to bladder (Edmondson et al., 2016).Although b3AR is also involved in lipolysis in human adipose(Bordicchia et al., 2014), the effects of b3AR activation in ratadipose appear to be much more profound compared with that

observed in human adipose, and this was noted in the earlyclinical programs looking at b3AR agonists for obesity (Ursinoet al., 2009). Chronic administration of b3AR agonists in obeserats results in weight loss without changes in food intake due toincreased energy expenditures (Ursino et al., 2009; Cerneckaet al., 2014), but no such effect is observed in humans (Arch,2008). b3AR is the predominant subtype in both white andbrown adipose tissue in rats, whereas in humans and rhesusmonkeys only in brown adipose does b3AR predominate, withlittle b3AR expression in white adipose (Candelore et al., 1999).This increased expression of b3AR in rat adipose and the morepronounced effect on metabolism may explain our observationof differential sensitivity to b3AR agonists in rat adipose versusrat bladder. Alternatively, the difference may not be in tissuesensitivity but may reflect physiologic differences in the rodentversus primate lower urinary tract, such that b3AR agonistsmay play more active of a role in adipose tissue in rats versusprimates.Our studies combining a b3AR agonist with antimuscarinics

resulted in enhanced bladder relaxation in both rats andrhesus monkeys, with greater synergism when both musca-rinic M2 and M3 subtypes were blocked compared withselective blockade of the M3 subtype. Although M3 appearsto be the predominant muscarinic subtype in mediatingcontractile responses in bladder detrusor muscle, the role ofM2 receptors in bladder is less certain despite it being themore highly expressed subtype (.90% in rat bladder) (Wanget al., 1995). It has been suggested that the M2 subtypecontributes to bladder relaxation but in a more indirect rolethrough the enhancement of M3-mediated contractions andthrough inhibition of bladder relaxation (Ehlert et al., 2005;Matsumoto et al., 2012). The inhibition of bladder relaxationmediated byM2 activation is thought to occur via an inhibitionof adenylyl cyclase (the M2 receptor subtype is Gi-coupled),which opposes the increase in cAMP elicited by activation ofb3AR (Matsui et al., 2003; Ehlert et al., 2007). In the absence ofan M2 response, forskolin and isoproterenol exhibit a greaterrelaxant activity compared with conditions under whichM2 isactive (Matsui et al., 2003; Ehlert et al., 2007). Inhibition ofM2activity would have a direct impact on the effects of b3ARactivation by increasing cAMP levels and would thereforeprovide greater relaxation mediated through b3AR activation,which is in accordance with our findings in this study.Recently, Furuta et al. (2016) reported that in consciousfemale rats, the combination therapy of a b3AR agonist andmuscarinic M3 antagonists was more effective in increasingbladder capacity than monotherapy and that M2 antagonismhad no impact on the effect of a b3AR agonist. We alsoconfirmed that simple M2 antagonism did not influenceb3AR agonist-induced relaxation of rat bladder strips. Addi-tionally, muscarinic receptor subtype contribution to bladder

Fig. 4. Effects of concentrations of a b3AR agonist CL316,243 andmuscarinic antagonists on EFS-induced contraction of rat bladder strips.(A) Representative recordings of isometric tension of bladder strips duringthe course of cumulative treatment of vehicle, CL316,243, and oxybutynin.(B) Dose titration of CL316,243, tolterodine, oxybutynin, and darifenacin.N = 7 or 8.

TABLE 3Potency of CL316,243, tolterodine, oxybutynin, and darifenacin in inhibiting EFS-induced contraction ofrat bladder strips and combination index of each antimuscarinic with CL316,243

Drug IC25 Upper 95% CI Lower 95% CI Combination Index with CL316,243

nM

CL316,243 2.86 5.91 1.18 —Tolterodine 4.71 11.68 1.89 0.21Oxybutynin 22.28 68.45 8.86 0.50Darifenacin 5.84 11.29 3.07 0.71

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function may differ between species. In nonhuman primates,muscarinic subtypes other thanM3 appear to contribute moreto bladder storage functions than rodents (Nagabukuro et al.,2011). Multiple clinical studies have demonstrated additionalbenefits by combining solifenacin and mirabegron (Abramset al., 2015; De Nunzio, et al., 2015; Kosilov et al., 2015).Because solifenacin is more M3 selective compared withtolterodine and oxybutynin (Ohtake et al., 2007), it may bepossible to improve further efficacy at bladder relaxationbased on our data, suggesting a b3AR agonist combined witha nonselective M2/M3 antagonist as the optimal combinationtherapy in humans.

In conclusion, we described the pharmacology of a newpotent and selective b3AR agonist, vibegron, and mechanisticinterplay between b3AR agonists and muscarinic antagonistsin urinary bladder. We also demonstrated that circulatingglycerol and free fatty acid levels could potentially be used assurrogate pharmacodynamic readouts to predict bladdereffects of vibegron, although care must be exercised whencomparing pharmacodynamic effects in different tissue beds(Morgan et al., 2012; Cook et al., 2014). The potential for b3ARagonists as monotherapy to effectively treat OAB has beenvalidated with the recent approval of mirabegron, but giventhat both muscarinic and b3ARs play a critical role in bladder

Fig. 5. Isobologram analysis of the effectsof combined treatments with a b3ARagonist CL316,234 and three differentmuscarinic antagonists in isolated ratdetrusor muscle: tolterodine (A), oxybuty-nin (B), and darifenacin (C). An additionalanalysis was done with darifenacin with apretreatment of methoctramine (D). Com-bination ratio of two drugs is shown inparentheses. Each represents mean 695% confidence interval.

TABLE 4Bladder capacity increases with combined treatments with vibegron and tolterodine and darifenacin in rhesus monkeysValues in table represent Mean 6 SEM of % increase over baseline (N=4).

VehicleVibegron

0.003 0.01 0.03 0.1 0.3 1

mg/kg

Vehicle 4.1 6 3.5 18.0 6 5.7 27.3 6 5.1 33.9 6 9.5 31.3 6 5.0 55.9 6 18.5Tolterodine 0.01 10.7 6 5.0a 26.7 6 4.9 31.0 6 6.3

0.03 16.8 6 13.9a 35.5 6 9.3 36.4 6 4.0 43.4 6 6.80.1 40.3 6 9.8a 62.9 6 8.3 56.8 6 8.1 69.6 6 14.6

Darifenacin 0.01 8.9 6 7.1a 13.7 6 4.0 23.7 6 3.30.03 18.3 6 8.6a 37.1 6 9.4 43.3 6 12.60.1 29.4 6 8.9a 58.0 6 19.5 68.7 6 20.9

aData from previous publication (Nagabukuro et al., 2011)


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function, additional efficacy (with potential for an improvedadverse effects profile) may be achieved by combining stan-dard of care antimuscarinics with b3AR agonists. Our obser-vations indicate that combination of b3AR agonists with dualM2 and M3 antagonists rather than selective M3 antagonistsprovides optimal efficacy in the treatment of OAB. Ourhypothetical translation to the clinic has yet to be determined,but we propose this potential therapeutic approach to redefinethe standard of care for the pharmacological treatment ofOAB.

Authorship Contributions

Participated in research design: Di Salvo, Nagabukuro, Wickham,Abbadie, DeMartino, Fitzmaurice, Gichuru, Donnelly, Jochnowitz,Hurley, Pereira, Sanfiz, Voronin, Villa, Woods, Zamlynny, Zycband,Salituro, Frenkl, Weber, Edmondson, and Struthers.

Conducted experiments: Di Salvo, Nagabukuro, Wickham, Abbadie,Fitzmaurice, Gichuru, Kulick, Donnelly, Jochnowitz, Hurley, PereiraSanfiz, Veronin, Villa Zamlynny, and Zycband.

Contributed new reagents or analytic tools: Di Salvo, Nagabukuro,Wickham, Abbadie, DeMartino, Fitzmaurice, Gichuru, Kulick,Donnelly, Jochnowitz, Hurley, Pereira Sanfiz, Veronin, Villa, WoodsZamlynny, Zycband, Salituro, Frenkl, Weber, Edmondson, andStruthers.

Performed data analysis: Di Salvo, Nagabukuro, Wickham,Abbadie, DeMartino, Fitzmaurice, Gichuru, Kulick, Donnelly,Jochnowitz, Hurley, Pereira, Sanfiz, Veronin, Villa, Woods Zamlynny,Zycband, Salituro, Frenkl, Weber, Edmondson, and Struthers.

Wrote or contributed to the writing of the manuscript: Di Salvo,Nagabukuro, Edmondson, and Struthers.

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Address correspondence to: Jerry Di Salvo, Evotec USA, 303B CollegeRoad East, Princeton, NJ 08540. E-mail: [email protected]


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